Antibiogram of Bacteria Isolated from Bloodstream Infection-Suspected Patients at the University of Gondar Comprehensive Specialized Hospital in Northwest Ethiopia: A Retrospective Study

Background Bacterial bloodstream infections (BSIs) are the leading cause of sepsis-related morbidity and mortality worldwide. The emergence and spread of antimicrobial resistance (AMR) in bacteria is also a growing global concern. As a result, data on bacterial profile and their antibiogram are essential for strategies to contain drug resistance, improve the quality of patient care, and strengthen health systems. Methods Retrospective data from bacteriological results of blood samples of BSI-suspected patients from 2018 to 2021 were collected using a data collection sheet. Standard bacteriological techniques were followed during sample collection, culture preparation, bacterial identification, and antibiotic susceptibility testing (AST). We used Epi Info version 7 to enter and clean the data and then exported it to SPSS version 26 for analysis. Logistic regression models were used to measure the association between variables. A p value <0.05 with a 95% confidence interval was considered as statistically significant. Result Of the total 2,795 blood culture records, 455 (16.3%) were culture positive for bacteria, with Klebsiella pneumoniae (26%) and Staphylococcus aureus (24.6%) being the leading isolates. The isolates were highly resistant to common antibiotics, with more than 80% of them being resistant to ceftriaxone and penicillin. Moreover, about 43% of isolates were multidrug resistant (MDR), with Klebsiella pneumoniae (65.5%), Acinetobacter species (56.7%), and Citrobacter species (53.8%) being the most common MDR isolates. Age and diagnosis year were significantly associated with the presence of bacterial BSIs (p value <0.05). Conclusion Bacterial BSI and AMR were growing concerns in the study area. Bacteremia was more common in children under the age of five, and it decreased as the patient's age increased. The alarming rate of AMR, such as MDR blood isolates, calls for periodic and continuous monitoring of antibiotic usage in the study area.


Introduction
Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response to infections, is the major global health threat with a high morbidity and mortality rate, particularly in sub-Saharan Africa (SSA) [1].In 2017, there were about 49 million sepsis-related incident cases, 41% of which occurred in children under the age of fve, and 11 million sepsis-related deaths, accounting for 20% of all annual deaths worldwide [2].Infections, particularly bacterial bloodstream infections (BSIs), have remained the leading cause of sepsis and sepsis-related mortality worldwide across all ages.Te presence of viable microorganisms in the bloodstream results in an infammatory response characterized by changes in clinical, laboratory, and hemodynamic parameters [3][4][5].Although the presence of viable bacteria in the bloodstream (bacteremia) is the commonly reported BSI worldwide, other organisms such as fungi, viruses, and parasites can also be involved [3].
In recent years, treating infections such as bacteremia has become a nightmare for the entire world due to the increasing resistance of the causative agents to most of the essential antimicrobials for human medicine [6].Indeed, one of the biggest threats to public health in the 21 st century is the emergence of antimicrobial resistance (AMR) in bacteria, which occurs when changes in bacteria lead to a reduction in the efectiveness of the drugs used to treat infections [7].Infections due to AMR bacteria result in death, longer duration of hospitalization, and pose a signifcant economic burden on national healthcare systems [8].For instance, in 2019, around 4.95 million deaths were associated with bacterial AMR, with the highest rate registered in low-resource settings, particularly SSA [9].Globally, the leading pathogens causing bacteremia are Escherichia coli (E.coli), Staphylococcus aureus (S. aureus), Klebsiella pneumoniae (K.pneumoniae), Pseudomonas aeruginosa (P.aeruginosa), Enterococcus species, Acinetobacter baumannii (A.baumannii), Coagulase-negative staphylococcus (CoNS), typhoidal and nontyphoidal Salmonella species, and others [10][11][12][13].At least fve of these bacteria are reported as the leading pathogens responsible for AMRrelated deaths in 2019 [9].
Te widespread and continuous emergence of AMR in bacteria causing BSIs coupled with the global spread of multidrug-resistant (MDR) strains is a major concern worldwide, particularly in low-and middle-income countries (LMICs) where testing coverage is low and healthcare systems are weak, which impacts AMR interventions [11,13].Given the global rise in BSIs and the evolving drug-resistance profles of the organisms involved, data on pathogen profles and their AMR patterns are essential for strategies to contain drug resistance, improve the quality of patient care, and strengthen health systems [11].Aside from the potential consequences of BSIs, delays in performing and receiving culture results, as well as the lack of susceptibility patterns for local isolates, lead to the frequent use of empirical therapy, which in turn contributes to the emergence of AMR, the consumption of expensive agents, and issues related to drug toxicity [13].As a result, identifying changes in pathogen distribution and AMR rates can help update diagnostic approaches, therapeutic strategies, and infection control and prevention measures in healthcare settings.

Study Setting, Design, and Period.
A retrospective crosssectional study was conducted at the University of Gondar Comprehensive Specialized Hospital (UoG-CSH) in northwest Ethiopia from May to June 2022.Te study involved the collection of data from patient culture records in the bacteriology laboratory of the UoG-CSH, which is a teaching and referral hospital located in Gondar City, 750 km northwest of Addis Ababa, Ethiopia.Tis hospital is one of the largest medical facilities in the country, with over 1,200 beds.Te hospital serves a population of more than 7 million people from Gondar and its surrounding areas.One of the notable features of the UoG-CSH is its accredited bacteriology laboratory, which plays a crucial role in the management of infectious diseases [14].

Study Population.
We included all patients who were suspected for BSIs and had recorded blood culture results registered on the bacteriology culture registration books from January 2018 to December 2021.Records with incomplete patient and laboratory data were excluded from the study.

Data Collection.
We conducted a retrospective review of four-year (January 2018 to December 2021) laboratory records of all blood cultures from patients suspected of having BSIs from all departments and units of the UoG-CSH.We used a data abstraction form to collect patients' sociodemographic and laboratory data (age, gender, diagnosis year, blood culture results, the isolated bacteria, and antimicrobial susceptibility testing (AST)) results from the laboratory record books.

Laboratory Methods.
Te UoG-CSH bacteriology laboratory used standard laboratory procedures to process blood culture and antimicrobial testing.Blood samples (10 mL for adults, 3 mL for children, and 1 mL for neonates) were collected by experienced healthcare workers from patients suspected of having BSIs using culture bottles with sterile tryptic soy broth (Oxoid Ltd., Basingstoke, UK).Te culture bottles were then incubated at 37 °C for 7 days and checked daily for signs of bacterial growth.Samples from culture bottles with signs of growth were then inoculated onto MacConkey agar, Blood agar, and Chocolate agar (BIO MARK Laboratories, India) and incubated for 24 hours.[15].Growth from these media was characterized, and species were identifed by a series of biochemical tests [16].Once the species were identifed, AST was carried out using Kirby-Bauer disc-difusion technique [17] on Muller-Hinton agar (MHA) (Oxoid Ltd., UK) following the Clinical Laboratory Standards Institute (CLSI) guidelines (2017-2020).Te bacterial suspension was standardized using 0.5 McFarland standard and inoculated on MHA (Oxoid Ltd., UK).Te antibiotic discs were dispensed after drying the plate for 3-5 min and incubated at 37 °C for 24 hours.Antibiotic discs such as penicillin ( 1).Age and diagnosis year were independently associated with the presence of bacterial BSI (p < 0.05).Te odds of neonates, children aged one to fve years, and infants having bacterial culture positive results were 3.4 (95% CI � 1.631-7.118,p � 0.001), 3.17 (95% CI � 2.063-4.877,p < 0.001), and 3.03 (95% CI � 1.990-4.617,p < 0.001) times higher, respectively, than adults aged 19 to 45 years.Regarding the year of diagnosis, the odds of bacterial culture positive results in 2021 and 2020 were 1.7 (95% CI � 1.242-2.321,p � 0.001) and 1.63 (95% CI � 1.144-2.326,p � 0.007) times higher, respectively, than in 2018 (Table 2).

Discussion
Bloodstream infections, mainly caused by MDR bacteria, are life-threatening events that require immediate interventions [19,20].Tus, this study identifed common bacteria and their AST pattern from BSI-suspected patients at the UoG-CSH.We found that bacteremia was a common healthcare problem in the study area, with under-fve children being the most afected.Te prevalence of bacteremia increased as the year progressed, with K. pneumoniae and S. aureus being the leading causative agents.Furthermore, more than 89% of the bacterial isolates were resistant to at least one antibiotic agent, including 92% of Gram-positive and 86.5% of Gramnegative isolates.We also found that about 43.5% of the bacterial isolates were MDR, with 56.7% of Gram-negative and 27.9% of Gram-positive isolates being MDR.Tese isolates were highly resistant to beta-lactam antibiotics such as penicillin and cephalosporins.

International Journal of Microbiology
Te overall prevalence of BSI in this study was 16.3% (95% CI � 14.9-17.6),which is comparable to reports in India (16.1%) [21] and Pakistan (16.6%) [22].In contrast, it is higher than reports in Ethiopia [23,24] and elsewhere [20,[25][26][27][28][29].However, previous retrospective studies from the same study area, including the years 2001 to 2005 [30], 2006-2012 [31], and 2012 to 2018 [14], reported a higher prevalence of BSI.Other studies in other parts of Ethiopia (Mekelle, 2015, and Addis Ababa, 2015-2016) [32,33] and elsewhere [34,35] also revealed a higher prevalence of culture-confrmed bacteremia.Te possible explanation for the variations of BSI prevalence across studies might be owing to diferences in the number of blood culture samples included, study population, methodology, study area, and infection prevention and control practices.We found a relatively higher prevalence of bacterial isolates in neonates (23.6%), followed by under-fve children (22.3%) and infants (21.3%), with a higher prevalence of S. aureus and K. pneumoniae.Tis fnding was in line with the study in Maldives, where neonates comprised a high proportion of BSI in comparison to infants and children [5].Similarly, a study in Bangladesh depicted that K. pneumoniae (75.96%) was the predominant isolate among neonates with sepsis [36].Similar to the current study, K. pneumoniae (40.4%) and S. aureus (17.0%) were identifed as the most common bacterial pathogens of BSI in children in Tanzania [37].Tis is an indication that the epidemiology of the previously leading bacterial etiologies of BSI has been changing possibly due to the implementation of vaccines and invasive treatment modalities [38,39].
We also found that bacterial BSI was higher in the neonatal and pediatric emergency ward, accounting for 23.4% and 20.2%, respectively.Tis fnding is comparable with previous reports (29.4% and 19.7%) from the same study area [40,41].However, it is higher than a report in Cameroon [42] and lower than reported in Rwanda [28].Te enrollment of large number of under-fve children in the current study may contribute to the discrepancies seen between studies, as children are more likely to expose to microorganisms as a result of contaminated food, soil, and other materials, as well as a lack of personal hygiene [43].Many studies showed that BSI is one of the most frequently observed infections in neonatal and pediatric wards.Te increased risk of sepsis due to bacteria is related to the persistent immune dysfunction [44].For example, in Europe, about 44.6% of healthcare-associated infections (HAIs) in children were reported as BSI, with a high prevalence in neonates and infants in their frst 11 months of life [45].Furthermore, infections caused by AMR bacterial pathogens are increasing in neonatal settings and infants admitted to the neonatal ward, particularly in those born preterm, with immature immunity, prolonged hospitalization, and frequent use of invasive medical devices [46].
Te results of our study showed that GNB were responsible for the majority of BSIs (54.3%), with K. pneumoniae (26.15%) being the most common cause, followed by S. aureus (24.62%),Enterococcus species (10.7%), and E. coli (9.67%).Comparable to this fnding, studies in Rwanda [28], India [34,47], and Ethiopia [41] reported K. pneumoniae, S. aureus, and E. coli as leading causes of BSIs.Infection prevention and control practices, geographic variation, medical environment, socioeconomic status, population, and prior use of inappropriate broad-spectrum antimicrobials may afect the distribution of bacterial pathogens [48,49].Te ability of K. pneumoniae to adapt the hospital environment, evade immune system, and resist antibiotics has led to an increase in its infection rate over the past few years [50].In K. pneumoniae, a variety of antibiotic resistance mechanisms can be involved, including changes in cell permeability, target gene mutation, plasmid-mediated resistance, and production of enzymes such as betalactamases, carbapenemase, and aminoglycoside-modifying enzymes [51,52].Additionally, the higher prevalence of K. pneumoniae and S. aureus in this study is possibly due to their predominance in the study area [53].
Gram-positive bacterial isolates in this study were highly resistant to penicillin (85.9%), piperacillin/tazobactam (83.3%), tetracycline (75.9%), ampicillin/amoxicillin (66.7%), and erythromycin (66.7%).Comparable to this fnding, a prospective study in Gondar reported a high resistance pattern of penicillin, ampicillin, and erythromycin in GPB [40].Tis could be related to the fact that these pathogens are known to have vast genetic repertoire to adapt and develop AMR, which indeed requires the global eforts in recognizing the emerging AMR mechanisms as to optimize the use of antibiotics and create strategies to circumvent this challenge [54].Gram-negative bacteria were also highly resistant to ceftriaxone (84.5%), oxacillin International Journal of Microbiology   International Journal of Microbiology   Te overall prevalence of MDR in this study was 43.5%, which is comparable to previous reports in Ethiopia [24,32,55].Among the total MDR isolates, 56.7% were GNB and 27.9% were GPB.Tis could be due to the increased presence of AMR determinants such as extended-spectrum beta lactamase (ESBL) and carbapenemase in GNB isolates in hospital settings [56,57].Furthermore, MDR prevalence was higher in K. pneumoniae (65.5%), followed by Acinetobacter spp.(56.7%) and Citrobacter spp.(53.8%).Te increasing trend of MDR may be due to the overuse and misuse of antibiotics, high load of infectious diseases, poor infection prevention and control practices, poor-quality antimicrobials, inadequate knowledge of AMR, misdiagnosis of etiologic agent, lack of advanced laboratories for AST, and poor hygiene and sanitation, especially in children [58][59][60].

Limitation of the Study.
Although this study reports the main bacteria involved in BSIs and their antibiotic susceptibility patterns, which is essential to rationalize the empiric antimicrobial therapy at the study area, we recognize that it has limitations.Te major limitation was attributable to the use of secondary data; due to the limited patient details recorded on the laboratory logbook, we could not access the full range of sociodemographic, behavioral, environmental, and clinical factors for bacterial BSI, which could afect the possible association that the factors may have with the presence of BSIs.Te availability of antibiotic disks for antibiotic susceptibility testing was not consistent over the four-year period, which may afect the analysis of antimicrobial susceptibility pattern of isolates.

Conclusion and Recommendation
Bacterial BSI is a growing concern in the study area, with a considerable increase in prevalence as the year progressed.Bacteremia was more common in children under the age of fve, particularly among neonates and infants, and reduced as the patient's age increased.Klebsiella pneumoniae and S. aureus were the leading causes of bacteremia, followed by Enterococcus spp.and E. coli.Tere was a high burden of AMR in the study area, with more than 80% of bacteremia-causing isolates becoming resistant to ceftriaxone and penicillin.Furthermore, more than half of Gram-negative isolates were MDR, with K. pneumoniae, Acinetobacter spp., and Citrobacter spp.being the leading MDR isolates.Terefore, routine bacterial identifcation and AST are recommended to detect the emergence and spread of AMR bacteria as early as possible.International Journal of Microbiology

Table 2 :
Factors associated with bacteremia at the UoG-CSH.

Table 1 :
Sociodemographic characteristics of patients at the UoG-CSH.
Figure 1: Distribution of bacterial isolates from blood culture of bacteremia-suspected patients at the UoG-CSH from January 2018 to December 2021, northwest Ethiopia.

Table 3 :
Distribution of bacteria recovered from blood samples with age category of bacteremia-suspected patients at the UoG-CSH.

Table 5 :
Antimicrobial susceptibility pattern of GNB isolated from bacteremia-suspected patients at the UoG-CSH.